Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Cell viability of K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days after doxycycline (Dox) addition (mean and SD, two-tailed Student’s t test, CENP-C WT cells: n = 6; CENP-C ΔM12BD cells: n = 6; ** p < 0.01). (B) Representative images of DAPI-stained K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Scale bar, 50 μm. (C) Population of normal interphase cells, mitotic cells, and cells with micronuclei in K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Error bars indicate SEM. n = 3 independent experiments; 200 cells from each cell line were quantified in each experiment. (D) The growth curve of K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. K562 WT or CENP-C ΔM12BD cells were treated with or without Dox ( KIF18A OFF or ON). The cell numbers were normalized to those at time 0 of each line. (E) Cell-cycle distribution of conditional knockout of KIF18A in K562 WT cells at each day after Dox addition, based on FACS analysis. (F) Cell-cycle distribution of conditional knockout of KIF18A in K562 CENP-C ΔM12BD cells at each day after Dox addition, based on FACS analysis. (G and H) Quantification of cells with misaligned chromosomes in K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. The experimental scheme is shown. Cells were stained with antibodies against MAD2 (red) to detect misaligned chromosomes and CENP-T (green) as a kinetochore marker. DNA was stained with DAPI (blue). Arrowheads show typical MAD2-positive unaligned chromosomes. Scale bar, 10 μm. The cells with MAD2-positive chromosomes were quantified (H) (mean and SEM, two-tailed Student’s t test; n = 5 independent experiments; n.s., non-significant; ** p < 0.01). (I) Numbers of MAD2 positive kinetochores in each cell in each condition (WT KIF18A ON; WT KIF18A OFF; CENP-C ΔM12BD KIF18A ON; CENP-C ΔM12BD KIF18A OFF) (Mean and SEM, n = 5 independent experiments).

Article Snippet: Mouse anti-human MAD2 , Santa Cruz Biotechnology , Cat#sc-65492; RRID: AB_831526.

Techniques: Knock-Out, Two Tailed Test, Staining, Marker

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A New Method for Chromosomes Preparation by ATP-Competitive Inhibitor SP600125 via Enhancement of Endomitosis in Fish

doi: 10.3389/fbioe.2020.606496

Figure Lengend Snippet: Effects of SP600125 on the expression levels of spindle assembly checkpoint genes. (A) qRT-PCR analysis for the differentially expressed BUB1, MAD2 , and CDC20 genes between the control group and the SP600125-treated cells. (B) Western blot analysis of expression levels of BUB1, MAD2, and CDC20 upon SP600125 treatment, where “1” represents the cells in the control group (without SP600125), “2” stands for the cells treated by SP600125 for 48 h, and “3” represents the cells first being treated by SP600125 for 48 h, then by SP600125-free culture for the next 12 h, and further by SP600125 treatment for another 48 h. Each bar represents the mean ± SD of three independent experiments (* P < 0.05).

Article Snippet: The membrane was blocked with 5% (w/v) non-fat milk in TBS containing 0.05% Tween-20 (TBST) for 1 h at 37°C and then incubated with primary antibodies, including anti-p53 (Abcam, USA), anti-MDM2 (Bioworld, USA), anti-p21 (Bioworld, USA), anti-Mad2L1 (Bioworld, USA), anti-CDC20 (Bioworld, USA), anti-BUB1 (Proteintech, USA), anti-Flag-tag (Proteintech, USA), and anti-β-ACTIN (Abcam, USA), overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A New Method for Chromosomes Preparation by ATP-Competitive Inhibitor SP600125 via Enhancement of Endomitosis in Fish

doi: 10.3389/fbioe.2020.606496

Figure Lengend Snippet: Analysis of cell proliferation by flow cytometry after transfection with pAd-MAD2. Lane 1 represents the normal cultured group. Lane 2 represents the negative control group. Lanes from 3 to 6 represent the overexpressed MAD2 groups (OE-MAD2): the cells being transfected with pAd-mCMV-MAD2-GFP-3-Flag-pA for 72 h (Lane 3), the cells being treated with SP600125 for the next 48 h (Lane 4), the cells continued to be further cultured in SP600125-free medium for 12 h (Lane 5), and the cells being further cultured using SP600125 treatment for another 48 h (Lane 6).

Article Snippet: The membrane was blocked with 5% (w/v) non-fat milk in TBS containing 0.05% Tween-20 (TBST) for 1 h at 37°C and then incubated with primary antibodies, including anti-p53 (Abcam, USA), anti-MDM2 (Bioworld, USA), anti-p21 (Bioworld, USA), anti-Mad2L1 (Bioworld, USA), anti-CDC20 (Bioworld, USA), anti-BUB1 (Proteintech, USA), anti-Flag-tag (Proteintech, USA), and anti-β-ACTIN (Abcam, USA), overnight.

Techniques: Flow Cytometry, Transfection, Cell Culture, Negative Control

Journal: Oncogene

Article Title: Mxi1-SRalpha: a novel Mxi1 isoform with enhanced transcriptional repression potential.

doi: 10.1038/sj.onc.1208107

Figure Lengend Snippet: Figure 1 The mxi1 gene can give rise to multiple mRNA transcripts that encode different protein isoforms. (a) The genomic organization of the mouse mxi1 locus (mxi1-gDNA) is shown. The exons (numbered 1–10) are represented by boxes, whereas the introns are represented by lines. Note that this locus is not drawn to scale, but double slashes through the introns between exons 1 and 2, 6 and 7, and 7 and 8 indicate the presence of large introns (18.7, 15, and 22.8 kb, respectively). (b) Three of the mouse mxi1 mRNA transcript forms resulting from alternative 50 exon usage are shown. Note that the mxi1-SRb form was known previously as mxi1-SR (Schreiber-Agus et al., 1995). (c) Schematic representation of the different protein isoforms capable of being encoded by the transcript forms depicted in (b). Note that the SRa and SRb forms are extended on their amino termini relative to the WR form, with the amino- acid sequences of these extensions shown below. The SRa extension can be subdivided into two portions: aa 1–61 represent a proline- rich domain of unknown function, while aa 62–103 are highly homologous to the SRb amino terminal extension and encode a putative SID (underlined). SID ¼ Sin3 interaction domain, BR ¼ basic region, HLH ¼ helix–loop–helix; CT ¼ carboxyl terminus, NT ¼ amino terminus. (d) Western blotting analysis of nuclear extracts and lysates from HeLa and 293T cells using the polyclonal anti-Mad2 (alternative name for Mxi1) antibody (Santa Cruz #1042). The MXI1-SRa band is indicated by a closed arrow, the MXI1-SRb band is indicated by an open arrow, and a nonspecific band migrating below the MXI1-SRb band is marked by an asterisk. The designation of the bands as the endogenous MXI1-SRa and MXI1-SRb proteins was further supported by the identical migration of transfected, nontagged versions of these proteins in 293T lysates (see transfection lanes for Mxi1-SRa and Mxi1-SRb). The specificity of the antibody was further validated by the altered migration of transfected MYC-tagged versions of Mxi1-SRa and Mxi1-SRb (right panel). Note that the endogenous MXI1-SRa band is enriched in nuclear extracts relative to lysates from both 293T and HeLa cells, while the endogenous MXI1-SRb band is detectable in nuclear extracts from 293T cells only. These results are consistent with those obtained on the RNA level by RT–PCR in which the MXI1-SRa transcript was readily detected in both HeLa and 293T cells and the MXI1-SRb in 293T cells only (data not shown). NE ¼ nuclear extract, lys ¼ lysate, NS ¼ nonspecific

Article Snippet: For detection of endogenous Mxi1-SRa and Mxi1-SRb proteins, total cell lysates or nuclear extracts (prepared as described in Kingston, 2004) from HeLa or 293T cells were fractionated by SDS–PAGE and probed with the MAD2 rabbit polyclonal antibody (Santa Cruz #sc-1042).

Techniques: Western Blot, Migration, Transfection, Reverse Transcription Polymerase Chain Reaction